This study was approved by the Animal Experiments Local Ethics Committee, Sakarya University, Turkey (01.07.2020: decision number: 34). Thirty-four 25-day-old prepubertal female Sprague-Dawley rats were included in the study. The number of rats was determined using G Power analysis (95% confidence interval, 80% Power). Rats were randomly divided into the propolis (n 17) and control groups (n 17). The weight of the rats was recorded before the experiments. The rats were sedated using anaesthetic doses of ketamine and xylazine; blood was obtained from the rats to measure the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol, and testosterone. Water-soluble propolis (1 cc at a dose of 200 mg/kg; based on other similar studies) was administered to the propolis group by gavage for 12 days (about 1 human year). Water-soluble propolis contains 10% pure propolis and had prepared using water and glycol solution. The content of propolis is presented in the supplementary table. The control group was administered 1 cc of water by gavage. The animals were provided food and water ad libitum. Vaginal openness was measured at baseline and then daily to determine the time of puberty onset. The number of rats attaining puberty after 12 days of treatment was recorded. After 12 days of treatment, the rats were weighed. Then, the rats were sedated with the appropriate anaesthetic dose, blood was obtained to measure the hormone levels, and the rats were sacrificed. Uterine, ovarian, and breast tissues were obtained for histopathological and immunohistochemical evaluation.
Histopathological and immunohistochemical evaluation
The tissues were fixed with 10% formalin solution for 48 h and dehydrated with 60%, 70%, 80%, 96%, and 100% alcohol. Then, the samples were passed through a xylol series to make the tissues transparent. The tissues were embedded in paraffin and cut using a microtome. The sections were stained with hematoxylin-eosin (HE) to observe the histological changes in the ovary, endometrium, and mammary gland tissues. Photographs were acquired under a light microscope (Olympus CX31-Japan). Ten sections (10 µm each) were obtained from each ovary to determine the effects of propolis on the number of follicles. Only follicles with oocyte nuclei were counted to determine the follicle count. Follicles were classified into five stages: primordial, primary, secondary, antral, and atretic follicles [14]. The automated image analysis software Image J® was used to measure endometrial thickness (µm). All slides were examined under the microscope at 100× magnification [15]. Mammary gland tissues were examined using a Nikon eclipse inverted microscope (Nikon Corp., Tokyo, Japan), and the area was calculated using the NIS-element imaging system from the same manufacturer. The ratio of the area of the secretory epithelium and fat cells to the area of the stroma was calculated (µm2) [16]. Ki-67 staining was used to demonstrate tissue stimulation and proliferation in the endometrium, mammary glands, and ovaries. The 4-µm-thick tissue samples were cut from the paraffin-embedded blocks and deparaffinized using a decreasing alcohol series. Citrate buffer was heated in the microwave for 20 min. The endogenous peroxidase activity was blocked with 3% H2O2. The primary antibody used was anti-Kİ-67 (1/400 dilution, Genetex). The secondary antibody (UltraVisionLarge Volume DetectionSystem Anti-rabbit by LabVision, HRP) was used in accordance with the manufacturer’s instructions. DAB has been used for immunohistochemical staining of secondary antibody-labeled proteins in tissues. Mayer's hematoxylin was used as a counterstain. The prepared slides were covered in mounting medium (Aqueous Mounting Medium by ScyTek). Proliferative activity, as assessed by Ki-67 staining, was semi-quantitatively analysed (h-score) by selecting 10 random fields, and 100 epithelial cells were photographed in each area. The Ki-67 index was calculated as the percentage of positively stained cells among the total cells assessed [17, 18] In the immunohistochemical analysis, Ki-67 staining and cell division rates in the mammary glands, ovary, and endometrium were compared between the control and propolis groups.
Hormonal assessment
The rats were sacrificed and blood samples collected. When the specimens had completely clotted, they were centrifuged at 1500 g for 10 min. Serum fractions were collected and frozen at −40°C until further use. LH, FSH, testosterone, and oestradiol levels were determined using a double antibody enzyme-linked immunosorbent assay (YLBiont brand Sandwich ELISA; Shanghai YL Biotech Co., Ltd., Shanghai, China). Hormone specific monoclonal antibody coated wells. Streptavidin–HRP-conjugated antibodies were added to all wells, except the blank well, and the wells were incubated at 37°C for 60 min. After incubation, the wells were washed to remove unbound antibody. The specimens were incubated with chromogen at 37°C for 10 min to develop a blue colour. Stop solution was added to terminate the reaction, reflected by a change in the colour of the solution from blue to yellow. The intensity of the yellow colour was directly proportional to the analyte concentration. The colorimetric readings were performed using the inappropriate wavelength for the micro ELISA reader. A standard curve was generated to calculate the sample concentrations. The results and the measurement range were specified as Rat LH 0.1-38 mIU/ml, Rat FSH 0.2-60 mIU/ml, Rat testosterone 10-3000 ng/L, Rat oestradiol 3-900 ng/L respectively. The within-run and between-run CV% of the analytes were given as <10%.
Statistical analysis was performed using the Statistical Package for the Social Sciences, version 20.0 software (IBM Inc., Chicago, IL, USA). Numerical variables were summarized by mean±standard deviation as appropriate. Normality of the numerical variables was assessed with the Kolmogorov–Smirnov test. To compare independent groups, the number of rats in the groups had low, nonparametric tests, including the Mann-Whitney U test. A p-value less than 0.05 was considered statistically significant.